PROJECT SUMMARY Nanopore sensing has been the focus of next-generation DNA sequencing technology. It has demonstrated great potential of rapid single-molecule DNA sequencing with long read lengths and simplified sample preparation. However, existing frameworks of nanopore preparation still face significant challenges from both the intrinsic resolution limit of ionic current detection and the engineering complications during fabrication, in order to achieve two-order-of-magnitude lower error rate and higher device yield/stability as required by human genome sequencing and clinical applications. Here we will address these challenges by exploring a unique fabrication framework combining top-down lithography and nanoscale electrochemistry to prepare solid-state nanopores that are self-embedded between a pair of transverse electrodes. This new scalable platform could allow precise control of DNA translocation and complimentary recognition tunneling readout, leading to more systematic DNA sequencing studies. This exploratory R21 project is based on our previous work on linearly tuning the size of a metal nanogap from 30 nm to 1 nm by electrodeposition, and our recognition tunneling sequencing studies. We hypothesize that: (1) the controlled electrodeposition process can be applied to a pair of sub-10 nm thick metal electrodes confined between two reservoir chambers, so that the gap can be precisely narrowed down into an ultra-thin tunneling junction, serving as the nanopore channel embedded between the electrodes; and (2) the control electrodes could enable gating the translocation of the DNA molecules by the transverse electric field, which also facilitates more reproducible recognition tunneling recording of different bases. To test our overall design and hypotheses, we will address two specific aims: (1) to develop robust scalable fabrication procedures and prepare prototype nanopore devices embedded within metal tunneling junctions, and (2) to explore effective control of DNA translocation using the integrated electrodes and investigate recognition tunneling readout for DNA sequencing with intrinsically higher resolution. We believe that our project has broad and translational significance, because the simple planar device layout, real-time fabrication control, and integration of control electrodes could enable reliable preparation of nanopore devices with well-controlled DNA translocation. Moreover, based on our recent results of fixed tunneling gap for reading DNA bases, the self-aligned nanopore and tunneling junction with the proper surface modification would allow more systematic investigation of recognition tunneling current readout with potentially higher bandwidth and better spatial resolution. Therefore our project can be developed into a novel framework that leads to large scale production of solid-state nanopore arrays for low-cost, high throughput sequencing, and serve as an affordable genomic tool for personalized medicine.